A Single-Molecule RNA Mobility Assay to Identify Proteins that Link RNAs to Molecular Motors

Methods Mol Biol. 2020:2166:269-282. doi: 10.1007/978-1-0716-0712-1_16.


mRNA transport and localization is a key aspect of posttranscriptional gene regulation. While the transport of many mRNAs is thought to occur through the recruitment of molecular motors, it has been a challenge to identify RNA-binding proteins (RBPs) that directly interact with motors by conventional assays. In order to identify RBPs and their specific domains that are responsible for recruiting a motor to transport granules, we have developed a single-molecule RNA mobility assay that enables quantifying the effect of a tethered RBP on the movement of an RNA. We demonstrate that tethering of RNAs to myosin or kinesin through their well-characterized interacting proteins results in quantitative differences in RNA mobility. This methodology provides a framework for identifying RBPs that mediate associations with motors.

Keywords: Kinesin; Live cell; Myosin; RNA transport; Single-molecule RNA imaging; Tethering assay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport, Active
  • Cell Line
  • Humans
  • Image Processing, Computer-Assisted / methods*
  • Kinesins / metabolism*
  • Levivirus / genetics
  • Luminescent Proteins
  • Microscopy, Confocal / methods*
  • Myosins / metabolism*
  • RNA, Messenger / metabolism*
  • RNA-Binding Proteins / metabolism*
  • Single Molecule Imaging / methods*


  • Luminescent Proteins
  • RNA, Messenger
  • RNA-Binding Proteins
  • Myosins
  • Kinesins